B cell lineage based immunogen design with humanized animals

ABSTRACT

Non-human animals with humanized immunoglobulin loci and methods of using them in vaccine design are described, as well as methods for making broadly neutralizing antibodies against infectious agents and pathogens are provided. Non-human animals with humanized immunoglobulin loci used in B-cell-lineage immunogen design in vaccine development are provided, as are methods of carrying out such design.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/174,563 filed Feb. 6, 2014, now U.S. Pat. No. 9,963,501, which claimsthe benefit under 35 USC § 119(e) of U.S. provisional application No.61/761,419 filed Feb. 6, 2013, which applications are hereinspecifically incorporated by reference in their entirety.

STATEMENT OF GOVERNMENT FUNDING

This invention was made with Government support under Grant No.UM1-A1100645, awarded by the National Institute of Health (NIH) andNational Institute of Allergy and Infectious Diseases (NIAID). TheGovernment has certain rights in this invention.

FIELD

Methods and compositions for immunogen design using B cell lineagedesign in non-human animals humanized immunoglobulin loci. Methods andcompositions for developing immunogens for making vaccines, employingnon-human animals with humanized immunoglobulin loci. Iterativeprocesses of priming and boosting of non-human animals having humanizedimmunoglobulin loci to identify candidate immunogens for use as vaccinesand for generating broadly neutralizing antibodies against infectiousagents and pathogens. Development of human vaccines in humanizedimmunoglobulin animals.

BACKGROUND

Vaccines against human pathogens are sorely needed, but difficult toobtain. Few, if any, suitable methods or systems are available forexposing a human immune system to a human infectious agent or pathogenand obtaining useful information that can be used to design a suitablevaccine that induces immunity in a human subject to the infectious agentor pathogen of interest. It is often not ethical, nor practical, toexpose human subjects to immunogens derived from a pathogen or aninfectious agent in a protocol for designing a vaccine, and non-humananimal systems are typically unsuitable for obtaining useful informationabout human immunogenicity.

Although humans can develop broadly neutralizing antibodies againstinfectious agents, and epitopes bound by such antibodies can beidentified, the epitopes are paradoxically relatively ineffective asimmunogens to induce immunity when used as vaccine candidates. This isbecause the epitopes recognized by broadly neutralizing antibodies arenot the same epitopes that bind early stage antibodies, such as thosepresent on early B cell receptors (BCRs) of the human immunoglobulinnaïve repertoire. Thus, vaccinating a human subject with an immunogenthat contains the epitope of a broadly neutralizing antibody may onlyrarely, if at all, result in any significant immunity to the infectiousagent in the human subject. That is to say, identifying an importantepitope that reacts with a broadly neutralizing antibody is, frequently,useless as a potential vaccine. This significant observation compels theconclusion that B cell clonal development toward mature B cells thatexpress broadly neutralizing antibodies is driven by immunogens that arenot identical to the epitope bound by the broadly neutralizing antibody.The problem, it seems, is complex and ties in with early B celldevelopment.

In order to develop a rational process of creating effective vaccinesfrom what is known of B cell development and the human immune responseto human infectious agents, a suitable system is needed for thedevelopment of candidate vaccines—a system that allows assessment of howthe human immune response interacts with immunogens and their variantsto ultimately develop a B cell line that produces a broadly neutralizingantibody to an epitope of a human infectious agent or pathogen, and—inthe process—to identify an immunogen variant that can serve as a vaccinethat can induce broadly neutralizing antibodies in the general humanpopulation. The process involves a search for a key immunogen that canprime the naïve human immunoglobulin repertoire and, with judiciouslyselected boost immunogens, drive the B cell clonal selection process toproduce a mature B cell that expresses broadly neutralizing antibodies.

There is a need in the art for compositions and methods for generatingvaccines that are suitable for immunizing humans against humaninfectious agents. There is a need in the art for rational methods fordesigning immunogens to be used as vaccines in human subjects. There isa need in the art for humanized systems, e.g., humanized animals, thatcan be used in methods to generate vaccines and vaccine candidates toimmunize humans against human infectious agents. There is a need for anon-human platform that can recapitulate a human B cell response, formaking vaccines to infectious agents by promoting development ofdesirable B cells through the use of immunogen variants that willultimately result in the development of desirable B cells that expressbroadly neutralizing antibodies against an infectious agent of interest.There is also a need to use humanized animals to further develop broadlyneutralizing antibodies to epitopes of infectious agents.

SUMMARY

Compositions and methods are provided for vaccine design using non-humananimals comprising humanized immunoglobulin sequences, includingiterative processes that comprise employing selected immunogens todevelop B cell clones from the naïve repertoire (e.g, immature B cellscomprising early B cell receptors) that mature into B cells that produceuseful antibodies that selectively bind particular immunogens, whereinone or more of the selected immunogens are employed as vaccines.Compositions and methods are described for generating immune cells (ingenetically modified non-human animals) that bind particular immunogensof interest, including developing immune cells from naïve or non-mutatedstates to mature or hypermutated states that specifically bind aninfectious agent of interest, and employing one or more immunogens ofinterest which achieved those steps in a vaccine preparation to developin a subject in need thereof an immune response to an infectious agentof interest, e.g., to develop an immunity in the subject of interest tothe infectious agent of interest, and immune cell populations thatexpress immunoglobulin sequences that bind antigens of interest. Methodsfor making vaccines using rodents with humanized immunoglobulinsequences are provided.

Compositions and methods are provided for facilitating B cell lineageimmunogen design using non-human animals having humanized immunoglobulinloci. Compositions and methods are provided for using the non-humananimals to identify, through intermediate ancestor B cells and B cellsbearing unmutated receptors (e.g., a naïve B cell repertoire, or a Bcell repertoire characterized by germline rearrangements that have notbeen mutated, or have not been somatically hypermutated), immunogens todevelop vaccines against infectious agents, which will elicit productionfrom a human immunoglobulin repertoire of mature B cells that expressbroadly neutralizing antibodies. Compositions and methods are providedfor prime and boost cycles using variants of immunogens and, optionally,adjuvants that generate broadly neutralizing antibodies, such thatimmunogen variants capable of stimulating an early or naïve human B cellrepertoire (e.g., a repertoire reflecting germline rearrangements) areidentified, wherein the immunogen variants induce the production of abroadly neutralizing antibody against the infectious immunogen.

General methods for making vaccines against human infectious agents andpathogens in non-human animals having humanized immunoglobulin loci areprovided.

Compositions and methods for using non-human animals having restrictedimmunoglobulin repertoires (e.g., a restricted human heavy chainrepertoire) are also provided.

In one aspect, methods and compositions are provided using B CellLineage-based immunogen design in non-human animals comprising ahumanized immunoglobulin locus (e.g., the VELOCIMMUNE® humanized mouse)for iterative development of vaccine immunogens for infectious diseases.

In one embodiment, infectious disease agent components are used asantigen-specific labels to identify precursors of protective antibodiesin unimmunized or uninfected VELOCIMMUNE® humanized mice or usingsimilar reagents and related strategies to identify immunogen-inducedclonal lineages of anti-infectious agent antibodies that are developingin the desired direction. These antibodies are then made to use astemplates for iterative vaccine design, wherein a series of novelimmunogens and, optionally, adjuvants to drive an otherwise subdominantor disfavored B cell maturation pathway to become dominant isdetermined. In one embodiment, sufficient plasma and tissue levels ofantibody are made to be protective in the setting of vaccination.

In one aspect, non-human animals comprising humanized immunoglobulinloci (e.g., VELOCIMMUNE® humanized mice) are used to design of vaccineimmunogens comprising: employing pre-immune unvaccinated mouse bonemarrow to isolate antigen-specific naïve B cells that bind toantigen-specific infectious agent reagents bearing broadly neutralizingepitopes; the antibodies reflective of the naïve B Cell Receptors (BCR)are isolated and made recombinantly; and immunogens are selected fortheir high affinity binding to these BCR in order to discover immunogensthat would selectively drive desired broadly neutralizing B celllineages to mature in peripheral lymph nodes and spleen.

In one aspect, non-human animals comprising humanized immunoglobulinloci (e.g., VELOCIMMUNE® humanized mice) are vaccinated with existingimmunogens that themselves are antigenic for broadly neutralizingantibodies one is trying to induce; the antibodies induced are thenisolated by making monoclonal antibodies from the spleens or lymph nodesof the immunized mice; the antibodies are made recombinantly; and thenthe most mature antibodies in the desired lineage used as a template tofurther drive the desired lineage to full expression of the desiredfunction, e.g., a protective effector function against an infectiousagent. In one embodiment, the protective function includes virusneutralization, antibody-dependent cellular cytotoxicity againstinfectious agent-infected cells, prevention of movement of infectiousagent across mucosal barriers, and blocking of entry of infectious agentin cell targets.

In one aspect, a method is provided for obtaining an immunogen thatresults in production in a non-human animal comprising a humanized Iglocus of a broadly neutralizing antibody against an infectious agent,comprising iterating and screening candidate immunogens for B celllineage vaccine design, wherein the iterating and screening comprises:

(a) identifying a first epitope that binds a broadly neutralizingantibody of an infectious agent;

(b) designing a second epitope that specifically binds a firstintermediate ancestor antibody of the broadly neutralizing antibody,wherein the second epitope is not identical to the first epitope;

(c) designing a third epitope that specifically binds a secondintermediate ancestor antibody of the broadly neutralizing antibody,wherein the third epitope is not identical to the second epitope; and,

(d) employing the third epitope as a vaccine against the infectiousagent.

In one embodiment, the method further comprises designing a fourthepitope that specifically binds a third intermediate ancestor antibodyof the broadly neutralizing antibody, wherein the fourth epitope isemployed as a vaccine against the infectious agent.

In one embodiment, the method further comprises designing a fifthepitope that specifically binds a fourth intermediate ancestor antibodyof the broadly neutralizing antibody, wherein the fifth epitope isemployed as a vaccine against the infectious agent.

In one embodiment, the first, second, third, fourth, or fifth epitopebinds an unmutated ancestor antibody. In one embodiment, the first,second, third, fourth, or fifth epitope that binds the unmutatedancestor antibody is employed as a vaccine against the infectious agent.

In one embodiment, the unmutated ancestor antibody is an antibody of anaive B cell. In one embodiment, the unmutated ancestor antibody isdisplayed on an early B cell as an IgM antibody. In one embodiment, theunmutated ancestor antibody comprises a rearrangement of germline genesegments prior to class switching.

In one aspect, a method for making a vaccine against an infectious agentin a non-human animal that comprises a humanized immunoglobulin locus isprovided, comprising priming and boosting with variants of an epitope ofthe infectious agent, wherein the epitope of the infectious agent bindsto a broadly neutralizing antibody. In one embodiment, the priming andboosting with variants of the epitope of the infectious agent iscontinued until a variant is found that, upon immunizing a naïvenon-human animal with a humanized Ig locus, the variant produces abroadly neutralizing antibody. In one embodiment, the variant thatproduces a broadly neutralizing antibody is employed as a vaccineagainst the infectious agent.

In one aspect, a method for designing a broadly neutralizing antibody(BNAb) in a non-human animal comprising a humanized immunoglobulin locusis provided, comprising exposing the animal to a first structuralvariant of an epitope of an immunogen that binds a broadly neutralizingantibody, allowing the animal to develop a first immune response to thefirst structural variant, and screening antibodies in the animal toobtain a broadly neutralizing antibody against the immunogen. In oneembodiment, the method comprises exposing the animal to a secondstructural variant of the epitope that binds the broadly neutralizingantibody, allowing the animal to develop a second immune response, andscreening antibodies from the animal to obtain a broadly neutralizingantibody against the immunogen, wherein the immunogen, the firststructural variant, and the second structural variant are not identical.

In one aspect, a method is provided for employing a non-human animalcomprising a humanized immunoglobulin locus in an algorithm to determinean unmutated ancestor antibody, comprising administering to thenon-human animal a first variant of an immunogen that binds a broadlyneutralizing antibody. In one embodiment, the algorithm employsquantitative inference based on the number of different VDJ combinations(roughly 10⁹) compared with the number of possible nucleotide sequencesof comparable length (roughly 4³⁵⁰). The algorithm infers back from thesequence of the broadly neutralizing antibody to the most plausibleancestor, or ancestors.

In one embodiment, the unmutated ancestral VDJ is determined by modelingthe somatic mutation rates, the probability that a given nucleotide willbe in a mutated states, and the number of passages through somatichypermutation. See, e.g., Haynes et al. (2012) B-cell-lineage immunogendesign in vaccine development with HIV-1 as a case study, NatureBiotech. 30(5):423-433 (hereby incorporated by reference, see, e.g.,especially Box 4).

In one aspect, a method is provided for employing a non-human animalwith a humanized immunoglobulin locus to optimize and validateimmunogens to develop a vaccine against an infectious agent, comprisingpriming and boosting the non-human animal with an immunogen of interestfor B cell lineage vaccine design. In one embodiment, the non-humananimal is primed and boosted with a first pre-selected immunogen, asecond pre-selected immunogen, and an nth pre-selected immunogen, untilthe non-human animal expresses a broadly neutralizing antibody againstthe infections agent. In one embodiment, the first pre-selectedimmunogen, the second pre-selected immunogen, or the nth pre-selectedimmunogen is an effective vaccine against the infectious agent. In oneembodiment, nth refers to third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth.

In one aspect, a method is provided for making a vaccine that elicitsproduction in a human subject of a broadly neutralizing antibody againstan infectious agent of interest, comprising priming and boosting anon-human animal comprising a humanized Ig locus with variants of anepitope of the infectious agent that binds a broadly neutralizingantibody, wherein a priming and boosting process employing variants ofthe epitope of the infectious agent that do not bind the broadlyneutralizing antibody, but produce in the non-human animal a broadlyneutralizing antibody that binds the infectious agent of interest.

In one aspect, a method is provided for making a vaccine that elicits ina human a broadly neutralizing antibody against an infectious agent ofinterest, comprising identifying an antibody that broadly neutralizesthe infectious agent of interest, making variants of the epitope boundby the broadly neutralizing antibody, performing prime and boost cycleswith variants of the epitope bound by the broadly neutralizing antibodyuntil an effective variant results in production of an antibody thatbroadly neutralizes the infectious agent of interest, and employing theeffective variant as a vaccine against the infectious agent of interest.

In one aspect, a method for B cell lineage-based vaccine design isprovided, comprising: (a) immunizing a non-human animal that comprises ahumanized immunoglobulin locus with a first immunogen of interest,wherein the first immunogen of interest comprises a plurality ofepitopes; (b) allowing the non-human animal to mount an immune responseto the first immunogen of interest, wherein the immune responsecomprises creating non-human B cells that express human immunoglobulinheavy chain VDJ and human immunoglobulin light chain VJ domains; (c)isolating clonally related memory B cells from the non-human animal thatspecifically bind a first epitope of interest on the first immunogen ofinterest; (d) determining VDJ and VJ sequence information for the VDJand VJ sequences of the memory B cells; (e) employing the determined VDJand VJ sequence information to determine an unmutated B cell receptor(BCR) VDJ and VJ sequence and, optionally, one or more intermediateancestor VDJ and VJ sequences between the BCR and the memory B cell;and, (f) making a second immunogen with enhanced affinity (as comparedwith the first immunogen) to the BCR and/or an intermediate ancestorantibody, wherein the second immunogen is employable in a human subjectas a vaccine against the first immunogen of interest.

In one embodiment, the first immunogen of interest is, or is derivedfrom, an infectious agent or a pathogen. In one embodiment, theinfectious agent or pathogen is a virus, a bacterium, a fungus, or aparasite. In one embodiment, the infectious agent is selected fromHIV-1, hepatitis C, dengue fever virus, and HPV.

In one embodiment, the first immunogen of interest is a protein or apeptide and the second immunogen of interest is a structural variant ofthe first immunogen of interest. In one embodiment, the structuralvariant comprises a substitution of one or more amino acids of theprotein or peptide. In one embodiment, the first immunogen is a proteinwith a postranslational modification (e.g., glycosylation, or presenceof a non-protein group, e.g., a lipid), and the second immunogen ofinterest is a variant of the postranslational modification.

In one embodiment, the VDJ is derived from a human V_(H)1-69 genesegment and a human D and a human J segment.

In one aspect, a method for designing a vaccine against an infectiousagent or pathogen is provided, comprising employing a non-human animalthat comprises a humanized immunoglobulin locus as a

In one aspect, broadly neutralizing antibodies of interest as usedherein are antibodies that exist in human subjects who are infected withan infectious agent of interest, wherein the antibodies are capable ofneutralizing the infectious agent in culture. In one embodiment, theantibodies are capable of preventing infection of a cell that saidinfectious agents can normally infect in the absence of the antibodies.

In various aspects, the infectious agent or pathogen is selected from avirus (e.g., human immunodeficiency virus (HIV), e.g., HIV-1, aninfluenza virus strain, human papillomavirus (HPV), hepatitis C,hepatitis B, hepatitis A, dengue), a bacterium, a fungus, a parasite(e.g., a malaria strain or species). etc.

In one aspect, a non-human animal comprising a humanized immunoglobulinlocus is provided, wherein the animal is immunized with a variant of anepitope of an infectious agent to which a first broadly neutralizinghuman antibody binds, and wherein the animal as the result of theimmunization with the epitope variant comprises a second broadlyneutralizing antibody that broadly neutralizes the same epitope of thesame infectious agent, wherein the second broadly neutralizing antibodyis not identical to the first broadly neutralizing antibody.

Compositions and methods are provided for identifying immunogens for useas vaccines against infectious agents, and for developing broadlyneutralizing antibodies against infectious agents, using non-humananimals comprising a humanized immunoglobulin locus that express a Bcell population derived from the entire human heavy chain variable generepertoire, operably linked to a plurality of human D segments and aplurality of human J segments; wherein the variable segments areoperably linked to a constant region, e.g., a human or non-humanconstant region on a transgene, or an endogenous non-human constantregion operably linked at the endogenous non-human locus to the humanheavy chain repertoire. In various embodiments the non-human animalcomprises a full and unrearranged human κ and/or λ light chain variablerepertoire, on a transgene linked to a human or rodent (e.g., mouse orrat) constant region gene, or at an endogenous non-human light chainlocus operably linked to a rodent (e.g., mouse or rat) constant regiongene.

Compositions and methods are provided for identifying immunogens for useas vaccines against infectious agents, and for developing broadlyneutralizing antibodies against infectious agents, using non-humananimals comprising a humanized immunoglobulin locus that express a Bcell population derived from no more than one, or no more than two,heavy chain variable region family members (e.g., V_(H)1-69 and/or itspolymorphs; V_(H)1-2 and/or its polymorphs) operably linked to aplurality of human D segments and a plurality of human J segments;wherein the variable segments are operably linked to a constant region,e.g., a human or non-human constant region on a transgene, or anendogenous non-human constant region operably linked at the endogenousnon-human locus to the restricted human V_(H) repertoire. In variousembodiments the non-human animal comprises a full and unrearranged humanκ and/or λ light chain variable repertoire, on a transgene linked to ahuman or rodent (e.g., mouse or rat) constant region gene, or at anendogenous non-human light chain locus operably linked to a rodent(e.g., mouse or rat) constant region gene.

Genetically modified non-human animals are provided that are immunizedwith human infectious agents, wherein the non-human animals compriseunrearranged human immunoglobulin variable region gene segments that arecapable of rearranging and forming genes that encode variable regionsequences operably linked to endogenous non-human constant regions,wherein the variable domains encoded by the sequences specifically bindthe infectious agent. In one embodiment, the non-human animal isimmunized with an immunogen that is a variant of an immunogen that bindsa broadly neutralizing human antibody, e.g., a broadly neutralizingantibody isolated from a patient infected with the infectious agent). Inone embodiment, the immunogen that the non-human animal is immunizedwith is not identical to the immunogen that binds the broadlyneutralizing antibody.

In one aspect, a non-human animal with a humanized immunoglobulin heavychain variable region gene locus and a humanized light chain variableregion gene locus is employed to screen for (or use in prime and boostcycles) immunogens to employ as vaccines against human infectious agentsor pathogens. In one embodiment, the immunogens are derived from, butare not identical to, immunogens that bind broadly neutralizingantibodies against the infectious agent or pathogen. In one embodiment,the identity of the screening immunogen is about 98%, 97%, 96%, 95%,90%, 85%, 80%, 75%, 70%, 65%, or 60% identical to the immunogen thatbinds the broadly neutralizing antibody.

In various aspects, the non-human animals comprising a humanizedimmunoglobulin locus comprises, e.g., an insertion of one or moreunrearranged human heavy chain V, D, and J and/or unrearranged humanlight chain V and J gene segments, either on a transgene (fully human orhuman variable/non-human constant) or operably linked to a non-humanconstant region at an endogenous non-human locus.

In various embodiments, the genetically modified heavy chain locuscomprises an endogenous Adam6a gene, Adam6b gene, or both, and thegenetic modification does not affect the expression and/or function ofthe endogenous Adam6a gene, Adam6b gene, or both.

In various embodiments, the non-human animal comprises an ectopicallypresent Adam6a gene, Adam6b gene, or both. In one embodiment, the Adam6agene is a non-human Adam6a gene. In one embodiment, the Adam6a gene is amouse Adam6a gene. In one embodiment, the Adam6a gene is a human Adam6agene. In one embodiment, the Adam6b gene is a non-human Adam6b gene. Inone embodiment, the Adam6b gene is a mouse Adam6b gene. In oneembodiment, the Adam6b gene is a human Adam6b gene.

In various embodiments, the one or more unrearranged human heavy chainV, D, and J and/or unrearranged human light chain V and J gene segmentsare present in a restricted repertoire, e.g., the number of human heavyand/or light gene segments present in the non-human animal is less thana complete human repertoire of the corresponding gene segments. Invarious aspects, the human gene segments are inserted at an endogenouslocus (directly or through a recombinase-containing cassette), andendogenous unrearranged gene segments are retained in the genome of thenon-human animal. In a specific embodiment, the human gene segments areinserted between the 3′-most non-human gene segment and the firstnon-human constant gene. In some embodiments where endogenous non-humanvariable gene segments are present, they are inactivated, e.g., by aninversion or a partial deletion.

In one aspect, the non-human animal is a rodent, e.g., a mouse or a rat.

In one aspect, the non-human animal comprises a plurality ofunrearranged human light chain V and J gene segments operably linked toa constant region (e.g., a human or non-human constant region, at anendogenous Ig locus or on a transgene), and the unrearranged human heavychain locus is restricted to no more than one, or no more than two, orno more than three V_(H) gene segments operably linked to one or morehuman D segments and one or more human J segments, at an endogenous Iglocus or on a transgene.

In one aspect, the non-human animal is a mouse or rat that comprises agermline modification resulting in no more than one, no more than two,or no more than three human V_(H) gene segments operably linked to aplurality of human D and a plurality of human J gene segments, whereinthe mouse or rat comprises a plurality of unrearranged human Vκ and Jκand/or human Vλ and Jλ gene segments operably linked to a human or anon-human constant region gene. In one embodiment, the plurality ofunrearranged human Vκ and Jκ and/or human Vλ and Jλ gene segments areoperably to endogenous constant region genes at an endogenous non-humanlight chain locus.

In one aspect, a method is provided for obtaining a broad spectrumantibody against an antigen of interest, comprising:

(a) priming a non-human animal by administering a first immunogenderived from the antigen of interest, wherein the non-human animalcomprises:

(i) a genetically modified immunoglobulin heavy chain locus comprisingone or more human V_(H) gene segments, a plurality of human D_(H) genesegments, and a plurality of human J segments, wherein the V_(H), D_(H),and J_(H) gene segments are operably linked to a heavy chain constantregion nucleic acid sequence,

(ii) a genetically modified immunoglobulin light chain locus comprisingone or more human V_(K) gene segments and one or more human J_(K) genesegments, wherein the V_(K) and the J_(K) gene segments are operablylinked to a light chain constant region nucleic acid sequence, and

wherein the first immunogen comprises a plurality of epitopes;

(b) allowing the non-human animal to mount an immune response againstthe first immunogen, wherein the immune response comprises generation ofnon-human B cells that express human immunoglobulin heavy chain (IgH)VDJ and human immunoglobulin light chain (IgL) VJ sequences;

(c) isolating clonally related B cells from the non-human animal thatexpress a B cell receptor (BCR) that specifically binds a first epitopeof the first immunogen;

(d) obtaining IgH VDJ and IgL VJ amino acid sequences expressed by the Bcell receptor (BCR) of the clonally related B cells;

(e) deducing from the IgH VDJ and IgL VJ sequences unmutated B cellreceptor (BCR) VDJ and VJ amino acid sequences, and one or moreintermediate ancestor B cell receptor (BCR) VDJ and VJ amino acidsequences expressed by B cells at an intermediate stage ofdifferentiation; and

(f) designing a plurality of immunogens that bind with enhanced affinityto the unmutated B cell receptor (BCR) or the intermediate ancestor Bcell receptor (BCR);

(g) boosting the immune response in the non-human animal byadministering to the non-human animal a second immunogen selected fromthe plurality of the immunogens in (f), wherein the second immunogencomprises a second epitope distinct from the first epitope; and

(h) obtaining the broad spectrum antibody from the non-human animal of(g).

In one embodiment, the immune response of the non-human animal isboosted by serially administering the plurality of immunogens identifiedin step (e) until the broad spectrum antibody is produced by thenon-human animal.

In one embodiment, the immune response of the non-human animal isboosted by administering a combination of the plurality of theimmunogens identified in step (e).

In one embodiment, the antigen of interest is, or is derived from, aninfectious agent or a pathogen, and the broad spectrum antibody is abroadly neutralizing antibody against the infectious agent or thepathogen.

In one embodiment, the infectious agent or pathogen is selected from thegroup consisting of a virus, a bacterium, a fungus, and a parasite.

In one embodiment, the infectious agent is selected from the groupconsisting of a human immunodeficiency virus (HIV), a hepatitis A virus,a hepatitis B virus, a hepatitis C virus, a dengue fever virus, and ahuman papillomavirus (HPV).

In one embodiment, all, or substantially all, functional endogenousV_(H) D_(H), and J_(H) gene segments in the immunoglobulin heavy chainlocus have been deleted or rendered non-functional, and wherein thegenetically modified immunoglobulin heavy chain locus comprises a singlehuman V_(H) gene segment or a polymorphic variant thereof, one or morehuman D_(H) gene segment, and one or more human J_(H) gene segment.

In one embodiment, the genetically modified heavy chain locus comprisesan endogenous Adam6a gene, Adam6b gene, or both, and the geneticmodification in the heavy chain locus does not affect the expression orfunction of the endogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the non-human animal comprises an ectopically presentAdam6a gene, Adam6b gene, or both.

In one embodiment, the single human V_(H) gene segment is selected fromthe group consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18,V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5,V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13,V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30,V_(H)3-30-3, V_(H)3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43,V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72,V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4- 30-1, V_(H)4-30-2,V_(H)4-30-4, V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61,V_(H)5-51, V_(H)6-1, V_(H)7- 4-1, V_(H)7-81 gene segment, and apolymorphic variant thereof.

In one embodiment, the single human V_(H) gene segment is a humanV_(H)1-69 gene segment or a polymorphic variant thereof.

In one embodiment, the single human V_(H) gene segment is a humanV_(H)1-2 gene segments or a polymorphic variant thereof.

In one embodiment, the single human V_(H) gene segment is a human V_(H)4-59 gene segment or a polymorphic variant thereof.

In one embodiment, the non-human animal is a rodent.

In one embodiment, the non-human animal is a mouse or a rat.

In one embodiment, the constant region nucleic acid sequence is a humanor a rodent nucleic acid sequence.

In one embodiment, the non-human animal is a mouse and the constantregion nucleic acid sequence is a mouse constant region nucleic acidsequence.

In one embodiment, all, or substantially all, functional endogenousV_(H) D_(H), and J_(H) gene segments in the immunoglobulin heavy chainlocus have been rendered non-functional, and the human V_(H), D_(H), andJ_(H) gene segments are present on a transgene.

In one embodiment, the human V_(H), D_(H), and J_(H) gene segments areoperably linked to an endogenous constant region nucleic acid sequenceat an endogenous non-human immunoglobulin heavy chain locus.

In one embodiment, the genetically modified immunoglobulin light chainlocus comprises a replacement of all, or substantially all, functionalV_(K) gene segments, J_(K) gene segments with one or more human V_(K)gene segments and one or more J_(K) gene segments, wherein the humanV_(K) gene segments and the J_(K) gene segments are operably linked to alight chain constant region nucleic acid sequence.

In one embodiment, the non-human animal comprises a replacement at theendogenous non-human immunoglobulin V_(K) locus of all or substantiallyall functional endogenous V_(K) gene segments with human V_(K) genesegments selected from V_(K)4-1, V_(K)5-2, V_(K)7-3, V_(K)2-4, V_(K)1-5,V_(K)1-6, V_(K)3-7, V_(K)1-8, V_(K)1-9, V_(K)2-10, V_(K)3-11, V_(K)1-12,V_(K)1-13, V_(K)2-14, V_(K)3-15, V_(K)1-16, V_(K)1-17, V_(K)2-18,V_(K)2-19, V_(K)3-20, V_(K)6-21, V_(K)1-22, V_(K)1-23, V_(K)2-24,V_(K)3-25, V_(K)2-26, V_(K)1-27, V_(K)2-28, V_(K)2-29, V_(K)2-30,V_(K)3-31, V_(K)1-32, V_(K)1-33, V_(K)3-34, V_(K)1-35, V_(K)2-36,V_(K)1-37, V_(K)2-38, V_(K)1-39, V_(K)2-40, and a combination thereof.

In one embodiment, the non-human animal comprises a replacement at theendogenous non-human immunoglobulin J_(K) locus of all or substantiallyall functional endogenous non-human immunoglobulin J_(K) gene segmentswith human J_(K) gene segments selected from J_(K)1, J_(K)2, J_(K)3,J_(K)4, J_(K)5, and a combination thereof.

In one embodiment, the one or more human V_(K) and J_(K) segments areoperably linked to an endogenous light chain constant region nucleicacid sequence at an endogenous non-human locus.

In various aspects, broad spectrum antibodies obtainable by any of themethods described herein are provided.

In various aspects, broadly neutralizing antibodies against aninfectious agent or a pathogen obtainable by any of the methodsdescribed herein are provided.

Further methods and compositions are described in the DetailedDescription herein, including but not limited to further descriptions ofB-cell-lineage immunogen design, and further descriptions of non-humananimals with humanized immunoglobulin loci, all of which can be used inconjunction with any of the aspects or embodiments described herein,unless otherwise indicated or unless the context dictates otherwise.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates general steps of the B cell lineage design-basedapproach to vaccine design. In the first step, the Ab1, Ab2, Ab3, andAb4 refer to four different antibody clonal lineages from peripheralblood memory B cells, one or more of which produces a protectiveantibody that is desired to be induced. Step 2 involves inference of anunmutated ancestor (UA) and intermediate (IA) antibodies in an antibodyclonal lineage. Step 3 involves suing the UA(s) and IAs as templates todesign immunogens with high affinity US and IA binding. This schematicillustrates a general scheme for regressing from an epitope of aninfectious agent that binds a mature, broadly neutralizing antibody toan unmutated ancestor B cell (e.g., UA) that binds a priming immunogen.Through B cell development the UA develops into an IA that binds anantigen for boosting, a process that is iterated until the maturebroadly neutralizing antibody is obtained. The iterative process allowsfor identification of suitable immunogens (antigens for prime andboosting) that may serve in the design of vaccines to elicit maturebroadly neutralizing antibodies. See, Haynes et al. (2012)B-cell-lineage immunogen design in vaccine development with HIV-1 as acase study, Nature Biotech. 30(5):423-433.

FIG. 2 lists two types of VELOCIMMUNE® humanized mice that can be usedfor this approach. Top panel: a VELOCIMMUNE® humanized mouse with areplacement of mouse endogenous sequences (closed symbols) with humangenomic sequences (closed symbols); Bottom panel: a V_(H) restrictedVELOCIMMUNE® humanized mouse with a single V_(H)1-9 gene segmentreplacing all endogenous mouse V_(H) gene segments, operably linked tohuman D and human J gene segments; for simplicity, the humanized κ locusis not shown for the Human V_(H)1-69 panel. Other V_(H) restrictedVELOCIMMUNE® humanized mice can similarly be used.

FIG. 3A shows normal B cell subsets of immune cells in VELOCIMMUNE®humanized mice in bone marrow and spleen, as compared with wild-typeBL/6 mice. FIG. 3B shows normal B cell subsets of immune cells inVELOCIMMUNE® humanized mice in peritoneum, as compared with wild-typeBL/6 mice.

FIG. 3C shows normal B cell subsets of immune cells in VELOCIMMUNE®humanized mice in Peyer's patch.

FIG. 4 shows normal T cell development and myeloid cell development ofimmune cells in VELOCIMMUNE® humanized mice. Top panel: BL/6 wild-typemice; Bottom panel: a V_(H) restricted VELOCIMMUNE® humanized mouse witha single V_(H)1-9 gene segment replacing all endogenous mouse V_(H) genesegments. T_(FH): B220⁻TCRβ⁺CD4⁺CXCR5⁺PD-1⁺Foxp3⁻; TFR:B220⁻TCRβ⁺CD4⁺CXCR5⁺PD-1⁺Foxp3⁺.

FIG. 5A shows plasma anti HIV-1 Envelope responses of VELOCIMMUNE®humanized mice to immunization with the HIV Envelope AE. A244 gp120protein (Alam, S. M. et al. (2013) Antigenicity and Immunogenicity ofRV144 Vaccine AIDSVAX Clade E Envelope Immunogen Is Enhanced by a gp120N-Terminal Deletion, J. Virol. 87: 1554-68).

FIG. 5B shows plasma anti HIV-1 Envelope responses of VELOCIMMUNE®humanized mice to immunization with the gp41 membrane proximal externalregion (MPER) liposome antigen. The plotted lines, from left to right,reflect Mouse RE506, Mouse RE506, Mouse RE507, and Mouse RE508.VELOCIMMUNE® humanized mice were immunized i.p. with MPER 656-GTH1liposomes/R848/oCpg/LipidA.

FIG. 6 shows a schema of the gp41 MPER liposome-peptide immunogen (tothe left) used in FIG. 5, containing the neutralizing epitopes for theHIV-1 broadly neutralizing antibodies 2F5 and 4E10 (Dennison, S. M. etal., (2009) Stable Docking of Neutralizing Human Immunodeficiency VirusType 1 gp41 Membrane-Proximal External Region Monoclonal Antibodies 2F5and 4E10 Is Dependent on the Membrane Immersion Depth of Their EpitopeRegions, J. Virol, 83:10211-10223). Top curve:

FIGS. 7A, 7B, and 7C show the ability to use a B cell tetramerreflective of the gp41 2F5 MPER neutralizing epitope (Verkoczy, L et al.(2009) Functional, Non-Clonal IgM^(a)-Restricted B Cell ReceptorInteractions with the HIV-1 Envelope gp41 Membrane Proximal ExternalRegion, PLoS One 4(10): e7215, 2009) to identify and isolate naïve Bcell precursors of this lineage from pre-immune VELOCIMMUNE® humanizedmice.

DETAILED DESCRIPTION Glossary

The term “broad spectrum antibody” as used herein includes antibodiesproduced by B cells that can bind a plurality of epitopes or antigenicdeterminants of an antigen. In one embodiment, the broad spectrumantibody possesses polyreactivity against various epitopes or antigenicdeterminants. In one embodiment, the broad spectrum antibody includesautoreactive antibodies expressed by ancestral B cells, immatureautoreactive B cells, or intermediate B cells.

The term “broadly neutralizing antibody” as used herein includesantibodies produced by B cells that neutralize diverse strains of aparticular infectious agent or a pathogen.

The term “the unmutated ancestor antibody” or “UA” as used hereinincludes antibodies expressed by a naïve B cell. In one embodiment, theunmutated ancestor antibody is displayed on an early B cell as an IgMantibody. In one embodiment, the unmutated ancestor antibody comprises arearrangement of germline gene segments prior to class switching.

The term “intermediate antibodies” or “IA” used herein includesantibodies made by intermediates in the clonal lineage generated byaffinity maturation of a naïve B cell in a germinal center.

The term “germinal center” as used herein includes a location in immunetissues at which dendritic and other cells present B cell contactantigen, helper T cells make contact with B cells, and immunoglobulinclass switching and somatic hypermutation take place.

Methods and compositions using B Cell Lineage Immunogen Design innon-human animals comprising a humanized immunoglobulin locus (e.g., theVELOCIMMUNE® humanized mouse) for iterative development of vaccineimmunogens for infectious diseases. This can be accomplished by use ofinfectious disease agent components as antigen-specific labels toidentify precursors of protective antibodies in unimmunized oruninfected VELOCIMMUNE® humanized mice or using similar reagents andrelated strategies to identify immunogen-induced clonal lineages ofanti-infectious agent antibodies that are developing in the desireddirection, and then producing these antibodies to use as templates foriterative vaccine design, an end goal of which is to identify a seriesof novel immunogens to drive an otherwise subdominant or disfavored Bcell maturation pathway to become dominant, and therefore producesufficient plasma and tissue levels of antibody to be protective in thesetting of vaccination.

A number of infectious diseases are characterized as having regions ofthe outer coats, envelopes or virion spikes to which neutralizingantibodies can be made, but for a variety of reasons, are not routinelymade. Examples of these infections include HIV-1, Hepatitis C, influenzaand dengue (reviewed in Haynes, B F et al. (2012) B-cell-lineageimmunogen design in vaccine development with HIV-1 as a case study,Nature Biotech 30:423-433). A strategy for overcoming many of theroad-blocks in induction of such protective anti-infectious agentantibodies has been proposed termed B Cell Lineage Immunogen Design, inwhich clonal lineages of rare desired antibodies are isolated,recombinantly expressed, and used as templates for vaccine design.

The VELOCIMMUNE® humanized mouse represents the most advanced form ofmice that carry humanized Ig loci. The full complement of human V_(H),Vκ, D, J_(H) and Jκ gene segments replace the endogenous loci.VELOCIMMUNE® humanized mice are capable of class-switch recombination,somatic hypermuation, and affinity-driven selection in germinalcenters—thus they are and excellent and unique model to rapidlyvaccinate or select pre-vaccination naïve precursors for use with B celllineage immunogen design to design novel immunogens for infectious agentvaccine development. VELOCIMMUNE® humanized mice that selectivelyexpress a limited V_(H) repertoire, or only one human V_(H) (such asV_(H)1-69 as just one representative example), can also advantageouslybe used for this purpose.

The VELOCIMMUNE® humanized mice can be used in two ways for design ofvaccine immunogens. First, pre-immune unvaccinated mouse bone marrow canbe used to isolate antigen-specific naïve B cells that bind toantigen-specific infectious agent reagents bearing broad neutralizingepitopes, the antibodies reflective of the naïve B Cell Receptors (BCR)isolated and made recombinantly, and then immunogens selected for theirhigh affinity binding to these BCR in order to discover immunogens thatwould selectively drive desired broad neutralizing B cell lineages tomature in peripheral lymph nodes and spleen. Second, VELOCIMMUNE®humanized mice can also be vaccinated with existing immunogens thatthemselves are antigenic for the broad neutralizing antibodies one istrying to induce, and the antibodies induced are then isolated by makingmonoclonal antibodies from the spleens or lymph nodes of the immunizedmice, the antibodies made recombinantly, and then the most matureantibodies in the desired lineage used as a template to further drivethe desired lineage to full expression of the desired function, e.g., aprotective effector function against an infectious agent. Thatprotective function can include virus neutralization, antibody-dependentcellular cytotoxicity against infectious agent-infected cells,prevention of movement of infectious agent across mucosal barriers, andblocking of entry of infectious agent in cell targets.

Thus, the described processes can identify immunogens by iterativedesign based on using VELOCIMMUNE® humanized mouse-derived antibodies astemplates for novel vaccine components.

Failed immunization of humans with human pathogen envelopes or epitopespresents a major public health challenge. Human immune systems are notalways able to develop broadly neutralizing antibodies against humanpathogens, including some of the most clinically important pathogens.Many reasons exist for these difficulties (reviewed in Haynes, B. F. etal. (2012) B-cell-lineage immunogen design in vaccine development withHIV-1 as a case study, Nature Biotech. 30(5):423-433). Whatever thereasons, it is clear that better and more useful systems formanipulation immune cells to develop more broadly neutralizingantibodies against clinically important antigens are in great need. Inparticular, powerful non-human systems for manipulating the developmentand design of B cell clones are in great need.

Because antibodies are ultimately derived from B cells that have(usually) undergone extensive selection, a detailed understanding of Bcell ontogeny, and crucial junctures in B cell development, arenecessary to develop useful clonal B cell lines that express broadlyneutralizing antibodies. Just as important as an understanding of thefine details of B cell ontogeny is the availability of a system that canbe manipulated to extract the benefits of our understanding of B cellontology. Rodents with humanized immunoglobulin loci, which generate Bcells that express antibodies with human sequences, may be useful.Currently, the best candidate system is the Regeneron VELOCIMMUNE®humanized mouse, which comprises a replacement at the endogenous mouseunrearranged heavy chain locus with the human unrearranged heavy chainlocus; and a replacement of the endogenous mouse unrearranged κ variablegene locus with the human unrearranged κ light chain locus; wherein thehuman unrearranged loci are operably linked to endogenous mouse constantregions. See, e.g., U.S. Pat. Nos. 7,105,348, 6,596,541, US Pat. Appl.Publ. Nos. 2007/0061900, 2011/0258710, 2011/0283376, 2012/0322108; eachhereby incorporated by reference. Non-human animals with restrictedheavy chain loci, e.g., V_(H)1-69 mice, may also be used; see, e.g.,U.S. Ser. No. 13/653,456, hereby incorporated by reference.

Compositions and methods are provided for developing vaccine immunogensusing animals that comprise humanized immunoglobulin loci. Non-humananimals (e.g., rodents, e.g., mice and rats) that are geneticallymodified to comprise human immunoglobulin variable domains and/or humanunrearranged heavy chain variable gene segments and unrearranged lightchain variable gene segments (e.g., κ, λ, or κ and λ) are used in aprocess to design vaccines, or vaccine candidates.

Varieties of Non-Human Animals with Humanized Immunoglobulin Loci

In various aspects, suitable humanized non-human animals for generatingB cells for use in the methods described herein include, for example,VELOCIMMUNE® humanized mice as described in, e.g., US 20012/0322108A1,US 2007/0061900A1, US 2011/0258710A1, US 2001/0283376A1, U.S. Pat. Nos.6,596,541, 7,105,248, each hereby incorporated by reference. Othersuitable non-human animals include, for example, non-human animalscomprising a limited heavy chain repertoire are described in U.S. Ser.No. 13/653,456, filed 17 Oct. 2012; as well as non-human animalsdisclosed in U.S. Ser. No. 61/658,466, filed 12 Jun. 2012, and U.S. Ser.No. 61/663,131, filed 22 Jun. 2012; each application and patentincorporated herein by reference.

Advantages of VELOCIMMUNE® humanized mice include that they exhibitnormal variable region usage and junctional diversity, normal somatichypermutation, normal numbers and distribution of B cells in spleen andlymph node, normal B cell differentiation in bone marrow, normal allelicexclusion, normal κ:λ light chain ratios, normal serum levels for all Igisotypes (IgM, IgG1, IgG2a, IgG2b, IgG3, IgE, IgA), normal immuneresponses to target antigens, and normal ability to generate stable,wherein “normal” refers to identical wild-type mouse littermates. Most,if not all, of these unique features of VELOCIMMMUNE® humanized mice areimportant for optimal B-cell-lineage immunogen design for vaccinedevelopment. VELOCIMMUNE® humanized mice (with restricted ornon-restricted heavy chain variable loci) are outstanding platforms forB-cell-lineage immunogen design for vaccine development at least in partbecause they are an excellent source of bone marrow B cell precursorsthat reflect the naïve human repertoire, a highly desired reagent forimmunogen design (see, e.g., FIG. 1, antibody UA (unmutated antibody),as well as an excellent source of intermediate ancestor antibodies (see,e.g., FIG. 1 at IA1, IA2, IA3), which can be readily primed and boostedto manipulate human variable domain-expressing intermediate ancestorantibodies.

The VELOCIMMUNE® humanized mice (those used herein without a restrictedrepertoire) comprise a replacement of endogenous mouse V, D, and J genesegments with the full human repertoire of functional V, D, and Jsegments, operably linked to the mouse heavy chain locus. TheVELOCIMMUNE® humanized mice (those used herein without a restrictedrepertoire) also comprise a replacement of endogenous mouse κ V and Jgene segments with the full human repertoire of functional human Vκ andJκ gene segments.

Non-Human Animals with Restricted Heavy Chain Repertoires: V_(H)1-69 andBeyond

VELOCIMMUNE® humanized mice with restricted repertoires, e.g., theV_(H)1-69 mouse used herein, comprises a replacement at the endogenousmouse locus of all functional mouse heavy chain V, D, and J segmentswith a single human V_(H)1-69 gene segment (present without polymorphs)operably linked to a plurality of human D segments and a plurality ofhuman J segments, which are operably linked to an endogenous mouseimmunoglobulin heavy chain locus.

The restricted heavy chain repertoire mouse is a useful tool in certaincircumstances, such as here, to generate clonal lines of B cells thatcan be used in immunogen design in vaccine development, for it iswell-known that certain human gene segments exhibit enhanced usage asagainst certain infectious agents or pathogens. V_(H)1-69-derived heavychains have been observed in a variety of antigen-specific antibodyrepertoires of therapeutic significance. For instance, V_(H)1-69 wasfrequently observed in heavy chain transcripts of an IgE repertoire ofperipheral blood lymphocytes in young children with atopic disease(Bando et al. (2004) Characterization of V_(H)E gene expressed in PBLfrom children with atopic diseases: detection of homologous V_(H)1-69derived transcripts from three unrelated patients, Immunology Letters94:99-106). V_(H)1-69-derived heavy chains with a high degree of somatichypermutation also occur in B cell lymphomas (Perez et al., (2009)Primary cutaneous B-cell lymphoma is associated with somaticallyhypermutated immunoglobulin variable genes and frequent use of V_(H)1-69and V_(H)4-59 segments, British Journal of Dermatology 162:611-618),whereas some V_(H)1-69-derived heavy chains with essentially germlinesequences (i.e., little to no somatic hypermutation) have been observedamong autoantibodies in patients with blood disorders (Pos et al. (2008)V_(H)1-69 germline encoded antibodies directed towards ADAMTS13 inpatients with acquired thrombotic thrombocytopenic purpura, Journal ofThrombosis and Haemostasis 7:421-428).

Further, neutralizing antibodies against viral antigens such as HIV,influenza and hepatitis C (HCV) have been found to utilize germlineand/or somatically mutated V_(H)1-69-derived sequences (Miklos et al.,(2000) Salivary gland mucosa-associated lymphoid tissue lymphomaimmunoglobulin V_(H) genes show frequent use of V_(H)1-69 withdistinctive CDR3 features, Blood 95(12):3878-3884; Kunert et al. (2004)Characterization of molecular features, antigen-binding, and in vitroproperties of IgG and IgM variants of 4E10, an anti-HIV type Ineutralizing monoclonal antibody, Aids Research and Human Retroviruses20(7):755-762; Chan et al. (2001) V_(H)1-69 gene is preferentially usedby hepatitis C virus-associated B cell lymphomas and by normal B cellsresponding to the E2 viral antigen, Blood 97(4):1023-1026; Carbonari etal., (2005) Hepatitis C virus drives the unconstrained monoclonalexpansion of V_(H)1-69-expressing memory B cells in type IIcryoglobulinemia: A model of infection-driven lymphomagenesis, Journalof Immunology 174:6532-6539; Wang and Palese (2009) Universal epitopesof influenza virus hemagglutinins?, Nature Structural & MolecularBiology 16(3):233-234; Sui et al. (2009) Structural and functional basesfor broad-spectrum neutralization of avian and human influenza Aviruses, Nature Structural & Molecular Biology 16(3):265-273; Marasca etal. (2001) Immunoglobulin Gene Mutations and Frequent Use of V_(H)1-69and V_(H)4-34 Segments in Hepatitis C Virus-Positive and Hepatitis CVirus-Negative Nodal Marginal Zone B-Cell Lymphoma, Am. J. Pathol.159(1):253-261).

However, these are just non-limiting examples of non-human animals thatcan be used as a platform for making B cell clones. Other humanizedrodents can be used to generate B cell populations as well, includingsystems that employ fully human trangenes or chimeric humanvariable/non-human constant transgenes, whether the non-human animals bemice, rats, or other non-human animals, e.g., other rodents. Further,suitable humanized non-human animals can comprise a full or a partialrepertoire of heavy and/or light chain gene segments. In variousaspects, all that is desired is the ability of the non-human animal todevelop a B cell clone expressing a variable domain that is broadlyneutralizing (or binds the pathogen of interest with sufficientlydesirable characteristics, e.g., high avidity, high specificity, etc.)that can be further developed to exhibit desired characteristics.

Thus, the non-human animals (e.g., rodents, e.g., mice or rats)described herein are useful in methods of vaccine design as againsthuman pathogens, based on B cell lineage design methods describedherein.

In one aspect, the non-human animal (e.g., a rodent, e.g., a mouse orrat) comprising a modified endogenous non-human immunoglobulin heavychain locus is provided, comprising a replacement of all functional Vgene segments with a single human V gene segment (or a single human Vgene segment present in multiple polymorphic forms or copy number),wherein the non-human immunoglobulin heavy chain locus is incapable ofrearrangement to form a heavy chain variable gene that is derived from aV gene segment other than the single human V gene segment (or one of thepolymorphic forms or copies).

In one embodiment, the single human V gene segment is V_(H)1-69. In oneembodiment, the single human V gene segment is V_(H)1-2.

In one embodiment, the locus comprises at least one human or non-humanD_(H) gene segment, and one human or non-human J_(H) gene segment. In aspecific embodiment, the locus comprises a human D_(H) gene segment anda human J_(H) gene segment. In a specific embodiment, the locuscomprises a human J_(H) gene segment. In another specific embodiment,the locus comprises a human V_(H)1-69 gene segment (present as a singlecopy or multiple copies of different polymorphic variants), allfunctional human D_(H) gene segments, and all functional human J_(H)gene segments. In another specific embodiment, the locus comprises ahuman V_(H)1-2 gene segment (present as a single copy or multiple copiesof different polymorphic forms), all functional human D_(H) genesegments, and all functional human J_(H) gene segments. In oneembodiment, the human V, D, and J gene segments (or V and J genesegments) are operably linked to a mouse constant region gene at anendogenous mouse heavy chain locus. In a specific embodiment, the mouseheavy chain locus comprises a wild-type repertoire of mouseimmunoglobulin constant region sequences.

In one aspect, a non-human animal (e.g., a rodent, e.g., a mouse or rat)comprising a modified immunoglobulin heavy chain locus is provided thatcomprises a heavy chain V segment repertoire that is restricted withrespect to the identity of the V segment, and that comprises one or moreD segments and one or more J segments, or one or more J segments. In oneembodiment, the heavy chain V segment is a human segment. In oneembodiment, the one or more D segments are human D segments. In oneembodiment, the one or more J segments are human J segments. In oneembodiment, the one or more D segments and one or more J segments arehuman D and human J segments.

In one embodiment, the modified locus is a non-human locus. In oneembodiment, the non-human locus is modified with at least one humanimmunoglobulin sequence.

In one embodiment, the restriction is to one V segment family member. Inone embodiment, the one V segment family member is present in two ormore copies. In one embodiment, the one V segment family member ispresent as two or more variants (e.g., two or more polymorphic forms ofthe V segment family member). In one embodiment, the one V segment is ahuman V segment family member. In one embodiment, the one V segmentfamily member is present in a number of variants as is observed in thehuman population with respect to that variant.

In one embodiment, the restriction is to a human V_(H)1-69 gene segment.In one embodiment, the human V_(H)1-69 gene segment is present in two ormore copies. In one embodiment, the human V_(H)1-69 gene segment ispresent as two or more variants (e.g., two or more polymorphic forms thehuman V_(H)1-69 gene). In one embodiment, the human V_(H)1-69 genesegment is present in a number of variants as is observed in the humanpopulation with respect to the human V_(H)1-69 gene segment.

In one embodiment, the restriction is to a human V_(H)1-2 gene segment.In one embodiment, the human V_(H)1-2 gene segment is present in two ormore copies. In one embodiment, the human V_(H)1-2 gene segment ispresent as two or more variants (e.g., two or more polymorphic forms thehuman V_(H)1-2 gene). In one embodiment, the human V_(H)1-2 gene segmentis present in a number of variants as is observed in the humanpopulation with respect to the human V_(H)1-2 gene segment.

In one aspect, a non-human animal comprising a humanized heavy chainvariable immunoglobulin locus is provided that comprises a singlefunctional human V segment. In one embodiment, the single functionalhuman V segment is selected from a V_(H)1-2, V_(H)1-3, V_(H)1-8,V_(H)1-18, V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69,V_(H)2-5, V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11,V_(H)3-13, V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3- 23,V_(H)3-30, V_(H)3-30-3, V_(H)3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38,V_(H)3-43, V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66,V_(H)3-72, V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1,V_(H)4-30-2, V_(H)4-30-4, V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59,V_(H)4-61, V_(H)5-51, V_(H)6-1, V_(H)7-4-1, and a V_(H)7-81 segment. Inone embodiment, the single functional human V segment is a V_(H)1-69segment; in a specific embodiment, the single functional human V segmentis present in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 polymorphicforms found in the human population. In one embodiment, the singlefunctional human V segment is a V_(H)1-2 segment; in a specificembodiment, the single functional human V segment is present in 1, 2, 3,4, or 5 polymorphic forms found in the human population.

In one aspect, a non-human animal comprising a restricted immunoglobulinheavy chain V gene segment repertoire is provided, wherein the non-humananimal further comprises one or more human immunoglobulin κ light chainvariable segments (Vκ). In one embodiment, the one or more Vκ segmentsare operably linked to one or more human J segments. In a specificembodiment, the J segments are human Jκ segments. In another specificembodiment, the non-human animal does not express an immunoglobulin λlight chain. In another specific embodiment, the non-human animal doesnot comprise a functional human or functional endogenous immunoglobulinλ light chain variable locus.

In one embodiment, the non-human animal is a rodent. In one embodiment,the rodent is selected from a mouse and a rat.

In one embodiment, the non-human animal comprises a replacement at theendogenous non-human immunoglobulin Vκ locus of all or substantially allfunctional endogenous Vκ segments with one or more functional human Vκsegments. In a further specific embodiment, the replacement is with allor substantially all functional human immunoglobulin Vκ segments.

In one embodiment, the non-human animal comprises a replacement at theendogenous non-human immunoglobulin Vκ locus of all or substantially allfunctional endogenous Vκ gene segments with human Vκ gene segmentsselected from Vκ4-1, Vκ5-2, Vκ7-3, Vκ2-4, Vκ1-5, Vκ1-6, Vκ3-7, Vκ1-8,Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15, Vκ1-16, Vκ1-17,Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26,Vκ1-27, Vκ2-28, Vκ2-29, Vκ2-30, Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35,Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, Vκ2-40, and a combination thereof.

In one embodiment, the non-human animal comprises a replacement at theendogenous non-human immunoglobulin Jκ locus of all or substantially allfunctional endogenous non-human immunoglobulin Jκ segments with one ormore functional human immunoglobulin Jκ segments. In a further specificembodiment, the replacement is with all or substantially all functionalhuman immunoglobulin Jκ segments.

In one embodiment, the non-human animal comprises a replacement at theendogenous non-human immunoglobulin Jκ locus of all or substantially allfunctional endogenous non-human immunoglobulin Jκ gene segments withhuman Jκ gene segments selected from Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and acombination thereof.

In a specific embodiment, the non-human animal comprises animmunoglobulin heavy chain variable region locus that comprises arepertoire of V segments consisting essentially of a single V segmentand/or polymorphic variants thereof. In one embodiment, the singleimmunoglobulin heavy chain V segment is a human V_(H)1-69 segment, andthe non-human animal further comprises a replacement of all functionalnon-human D_(H) segments with all functional human D_(H) segments, andfurther comprises a replacement of all functional non-human J_(H)segments with all functional human J_(H) segments, and wherein theimmunoglobulin heavy chain variable region locus is operably linked to ahuman or non-human constant region gene sequence. In a specificembodiment, the constant region gene sequence is an endogenous non-humanconstant region gene sequence. In a specific embodiment, the non-humananimal rearranges segments at the non-human immunoglobulin heavy chainlocus to form a gene encoding heavy chain variable region comprising ahuman V_(H)1-69 sequence, a human D_(H) sequence, a human J_(H)sequence, and a mouse constant region sequence.

In a specific embodiment, the non-human animal comprises animmunoglobulin heavy chain variable region locus that comprises arepertoire of V segments consisting essentially of a single V segmentand/or polymorphic variants thereof. In one embodiment, the singleimmunoglobulin heavy chain V segment is a human V_(H)1-2 segment, andthe non-human animal further comprises a replacement of all functionalnon-human D_(H) segments with all functional human D_(H) segments, andfurther comprises a replacement of all functional non-human J_(H)segments with all functional human J_(H) segments, and wherein theimmunoglobulin heavy chain variable region locus is operably linked to ahuman or non-human constant region gene sequence. In a specificembodiment, the constant region gene sequence is an endogenous non-humanconstant region gene sequence. In a specific embodiment, the non-humananimal rearranges segments at the non-human immunoglobulin heavy chainlocus to form a gene encoding heavy chain variable region comprising ahuman V_(H)1-2 sequence, a human D_(H) sequence, a human J_(H) sequence,and a mouse constant region sequence.

In one aspect, a non-human animal comprising a restricted immunoglobulinheavy chain V segment repertoire is provided, wherein the non-humananimal comprises one or more human λ light chain variable (Vλ) segments.In one embodiment, the one or more human Vλ segments are operably linkedto one or more human J segments. In a specific embodiment, the Jsegments are human Jλ segments. In another specific embodiment, thenon-human animal does not express a κ light chain. In another specificembodiment, the non-human animal does not comprise a functional human ornon-human κ light chain variable locus.

In one embodiment, the non-human animal further comprises a replacementof all or substantially all functional non-human immunoglobulin

In one embodiment, the non-human animal comprises a replacement of allor substantially all functional non-human immunoglobulin Vλ segmentswith one or more functional human immunoglobulin Vλ segments. In afurther specific embodiment, the replacement is with all orsubstantially all functional human immunoglobulin Vλ segments.

In one embodiment, the non-human animal comprises a replacement of allor substantially all functional non-human Vλ segments with a fragment ofcluster A of the human λ light chain locus. In a specific embodiment,the fragment of cluster A of the human λ light chain locus compriseshuman Vλ gene segments Vλ 3-27 through Vλ 3-1.

In one embodiment, the non-human animal comprises a replacement of allor substantially all functional non-human Vλ segments with a fragment ofcluster B of the human λ light chain locus. In a specific embodiment,the fragment of cluster B of the human λ light chain locus compriseshuman Vλ gene segments Vλ 5-52 through Vλ 1-40.

In one embodiment, the non-human animal comprises a replacement of allor substantially all functional non-human Vλ segments with a fragment ofcluster A and a fragment of cluster B of the human λ light chain locus,wherein as a result of the replacement comprise human Vλ gene segmentsVλ 5-52 through Vλ 3-1.

In one embodiment, the non-human animal comprises a replacement of allor substantially all functional non-human Vλ segments with at least 12human Vλ gene segments, at least 28 human Vλ gene segments, or at least40 human Vλ gene segments.

In one embodiment, the non-human animal comprises a replacement of allor substantially all functional non-human immunoglobulin Jλ genesegments with one or more functional human immunoglobulin Jλ genesegments. In a further specific embodiment, the replacement is with allor substantially all functional human immunoglobulin Jλ gene segments.In various embodiments, the functional human Jλ gene segments includeJλ1, Jλ2, Jλ3 and Jλ7.

In a specific embodiment, the non-human animal comprises animmunoglobulin heavy chain variable (V_(H)) region locus that comprisesonly a single V_(H) segment, wherein the single V_(H) segment is a humanV_(H)1-69 segment or a human V_(H)1-2 segment, and further comprises areplacement of all functional non-human D_(H) segments with allfunctional human D_(H) segments, and further comprises a replacement ofall functional non-human J_(H) segments with all functional human J_(H)segments, and wherein the V_(H) region locus is operably linked to ahuman or non-human constant region gene sequence. In a specificembodiment, the constant region gene sequence is a non-human constantregion gene sequence, e.g., an endogenous non-human constant genesequence. In a specific embodiment, the non-human animal rearrangessegments at the non-human immunoglobulin heavy chain locus to form agene encoding an immunoglobulin heavy chain variable region comprising ahuman V_(H)1-69 sequence (or a human V_(H)1-2 sequence), a human D_(H)sequence, a human J_(H) sequence, and an endogenous non-human constantregion sequence.

We claim:
 1. A method for screening for a broad spectrum antibodyagainst an antigen of interest, comprising: (a) administering a firstimmunogen derived from the antigen of interest to a mouse from a mouseline comprising in its germline: (i) a restricted immunoglobulin heavychain locus comprising a single human unrearranged V_(H) gene segment,one or more human D_(H) gene segments, and one or more human J_(H) genesegments operably linked to a heavy chain constant region nucleic acidsequence, (ii) a genetically modified immunoglobulin light chain locuscomprising one or more human V_(K) gene segments and one or more humanJ_(K) gene segments, wherein the V_(K) and the J_(K) gene segments areoperably linked to a light chain constant region nucleic acid sequence,and wherein the first immunogen comprises a plurality of epitopes; (b)allowing the mouse to mount an immune response against the firstimmunogen, wherein the immune response comprises generation of mouse Bcells that express human immunoglobulin heavy chain (IgH) VDJ and humanimmunoglobulin light chain (IgL) VJ sequences; (c) isolating clonallyrelated B cells from the mouse that express a B cell receptor (BCR) thatspecifically binds a first epitope of the first immunogen; (d)determining IgH VDJ and IgL VJ amino acid sequences of the B cellreceptor (BCR) of the clonally related B cells; (e) deducing from theIgH VDJ and IgL VJ sequences unmutated B cell receptor (BCR) VDJ and VJamino acid sequences; and one or more intermediate ancestor B cellreceptor (BCR) VDJ and VJ amino acid sequences of the B cells at anintermediate stage of differentiation; (f) providing a plurality ofsecond immunogens comprising second epitopes distinct from the firstepitope that bind with enhanced affinity to the unmutated B cellreceptor (BCR) or the intermediate ancestor B cell receptor (BCR)relative to the first epitope of the first immunogen; (g) seriallyadministering to another mouse of the mouse line second immunogensselected from the plurality of the second immunogens in (f), the serialadministration starting with a second immunogen to the unmutated BCR andfollowing with second immunogens to intermediate BCRs successively moredistant phylogenically from the unmutated BCR; and (h) determiningwhether the immune response from the mouse of (g) includes a broadspectrum antibody, which binds a plurality of the second epitopes. 2.The method of claim 1, wherein the first and second immunogens of steps(a) and (g) are administered as a liposomal complex.
 3. The method ofclaim 1, wherein the antigen of interest is, or is derived from, aninfectious agent or a pathogen, and the broad spectrum antibody is abroadly neutralizing antibody against the infectious agent or thepathogen.
 4. The method of claim 1, wherein all, or substantially all,functional endogenous V_(H) D_(H), and J_(H) gene segments in theimmunoglobulin heavy chain locus have been deleted or renderednon-functional.
 5. The method of claim 4, wherein the restrictedimmunoglobulin heavy chain locus comprises an endogenous Adam6a gene,Adam6b gene, or both, and the genetic modification in the heavy chainlocus does not affect the expression or function of the endogenousAdam6a gene, Adam6b gene, or both.
 6. The method of claim 4, wherein themouse comprises an ectopically present Adam6a gene, Adam6b gene, orboth.
 7. The method of claim 1, wherein the heavy or light chainconstant region nucleic acid sequence is a human or a rodent nucleicacid sequence.
 8. The method of claim 1, wherein the heavy or lightchain constant region nucleic acid sequence is a mouse constant regionnucleic acid sequence.
 9. The method of claim 1, wherein all, orsubstantially all, functional endogenous V_(H) D_(H), and J_(H) genesegments in the immunoglobulin heavy chain locus have been renderednon-functional, and the human V_(H), D_(H), and J_(H) gene segments arepresent on a transgene.
 10. The method of claim 1, where the humanV_(H), D_(H), and J_(H) gene segments are operably linked to anendogenous constant region nucleic acid sequence at an endogenousnon-human immunoglobulin heavy chain locus.
 11. The method of claim 1,wherein the genetically modified immunoglobulin light chain locuscomprises a replacement of all, or substantially all, functionalendogenous V_(K) gene segments and J_(K) gene segments with one or morehuman V_(K) gene segments and one or more human J_(K) gene segments,wherein the human V_(K) gene segments and the human J_(K) gene segmentsare operably linked to a light chain constant region nucleic acidsequence.
 12. The method of claim 1, wherein the mouse comprises areplacement at the endogenous non-human immunoglobulin V_(K) locus ofall or substantially all functional endogenous V_(K) gene segments withhuman V_(K) gene segments selected from the group consisting ofV_(K)4-1, V_(K)5-2, V_(K)2-4, V_(K)1-5, V_(K)1-6, V_(K)1-8, V_(K)1-9,V_(K)2-10, V_(K)3-11, V_(K)1-12, V_(K)1-13, V_(K)2-14, V_(K)3-15,V_(K)1-16, V_(K)1- 17, V_(K)2-18, V_(K)2-19, V_(K)3-20, V_(K)6-21,V_(K)1-22, V_(K)1-23, V_(K)2-24, V_(K)3-25, V_(K)2-26, V_(K)1-27,V_(K)2-28, V_(K)2-29, V_(K)2-30, V_(K)3-31, V_(K)1-32, V_(K)1-33,V_(K)3-34, V_(K)1-35, V_(K)2-36, V_(K)1-37, V_(K)2-38, V_(K)1-39,V_(K)2-40, and a combination thereof.
 13. The method of claim 1, whereinthe mouse comprises a replacement at the endogenous non-humanimmunoglobulin J_(K) locus of all or substantially all functionalendogenous non-human immunoglobulin J_(K) gene segments with human J_(K)gene segments selected from the groups consisting of J_(K)1, J_(K)2,J_(K)3, J_(K)4, J_(K)5, and a combination thereof.
 14. The method ofclaim 11, wherein the one or more human V_(K) and J_(K) segments areoperably linked to an endogenous light chain constant region nucleicacid sequence at an endogenous non-human locus.
 15. The method of claim1, wherein the antigen of interest is from a pathogen.
 16. The method ofclaim 15, wherein the pathogen is HIV, influenza virus or hepatitis Cvirus.
 17. The method of claim 1, wherein the single human unrearrangedV_(H) gene segment is a human V_(H)1-69 gene segment or a polymorphicvariant thereof.
 18. The method of claim 15, wherein the single humanunrearranged V_(H) gene segment is a human V_(H)1-69 gene segment or apolymorphic variant thereof.